Journal of Experimental Microbiology and Immunology (JEMI) Vol. 13:89-92 Copyright © April 2009, M&I UBC The Reduction of T7 Phage Adsorption in Escherichia coli B23 Cells Treated with Sub-Lethal Levels of Kanamycin Joanne Bleackley, Jesse Cooper, Monica Kaminski and Stephanie Sandilands Department of Microbiology & Immunology, UBC The production of capsular material has been implicated as a bacterial defence mechanism against certain antibiotics and against phage adsorption. The capsule acts to block the phage from binding to its receptor on the bacterial cell which effectively inhibits phage adsorption. Previously, it has been shown that exposing Escherichia coli B23 cells to sub-inhibitory levels of the antibiotic kanamycin results in an increase in extracellular polysaccharide production, possibly in the form of a capsule. In the current study, this phenomenon was investigated using T7 phage adsorption as a model to assess the presence of a capsule. Phage adsorption assays were performed on both E. coli B23 cells treated with sub-lethal levels of kanamycin for 1 hour and untreated cells. T7 phage adsorption was shown to be diminished in E. coli treated with antibiotic, as compared to those which had not been treated with antibiotic. This decrease in phage adsorption to the antibiotic treated cells suggests that the induced extracellular polysaccharide produced by these cells is part of the capsule. Thus, this work provides further insight into the likely nature of the induced-extracellular polysaccharide produced by these cells following antibiotic treatment. Bacteria have developed mechanisms to adapt to phage adsorption (17). Binding of T7 phage to LPS changes in their environment. The production of can also be blocked by capsular molecules, including extracellular polysaccharides is a means to preserve colonic acid. Colanic acid is one of the major bacteria in their natural habitats by excluding components of the capsule produced by E. coli B23 in potentially harmful compounds (18). These response to hostile environments; implicating colanic extracellular polysaccharides are often assembled in a acid synthesis as a survival mechanism in damaged capsular formation, thus forming a physical barrier to cells (13). It has been shown that sub-inhibitory the environment (13). Specifically, this protective layer concentrations of certain antibiotics, such as β-lactams, is capable of shielding the cell from antibiotics, induce expression of the capsular polysaccharide amoebic attack, desiccation, phagocytosis and operon (cps) which encodes genes required for colanic bacteriophage infection (18). Capsules interfere with acid synthesis (13). phage adsorption by either covering the receptor Previous groups investigating the effect of sub-lethal completely, or by interfering with receptor-ligand levels of aminoglycoside antibiotics on E. coli, have binding. In order for infection to occur, the phage also reported an increase in extracellular polysaccharide must be able to inject its DNA into the cytoplasm of the production (3, 5, 9). Aminoglycosides, such as bacteria. Therefore if the thickness of the capsule is kanamycin, act within the bacterial cell to irreversibly greater than the length of the phage tail, the bacteria bind the 30S subunit of the ribosome effectively will be impermeable to phage infection. In this regard, inhibiting protein translation (9). Their effect on the capsular production acts as a non-specific phage transcription of cps operon has not yet been elucidated. inhibitor. Previous studies have shown that capsule An experiment performed by Lu et al., involving the thickness and phage resistance are proportional (18). exposure of E. coli B23 to sub-inhibitory levels of The negative charge of the capsule is also an important kanamycin, found a significant increase in extracellular factor in the resistance to phage adsorption (18). polysaccharide production (9). They theorized that this The T7 phage adsorption system involves the increase was due to the production of a capsule in the interaction between the tail fibre protein of T7 phage antibiotic-exposed bacteria. To test this hypothesis we and lipopolysaccharide (LPS) on the surface of used the T7 phage- adsorption system to determine if Escherichia coli B23 (14). E. coli strains that are antibiotic treated cells would have reduced phage deficient in LPS, or have mutations in the heptose adsorption thus indicating increased capsule. moiety of LPS, have been shown to be resistant to T7 89 Journal of Experimental Microbiology and Immunology (JEMI) Vol. 13:89-92 Copyright © April 2009, M&I UBC MATERIALS AND METHODS RESULTS Phage preparation. A stock solution of T7 phage and Escherichia Phage Adsorption Assay. The percentage of coli B23 culture were obtained from the MICB 421 culture collection in the Department of Microbiology and Immunology at the University adsorbed phage after 4 and 7 minutes of incubation of British Columbia. To enumerate phage, an overnight culture was with the untreated cells during the phage adsorption prepared by inoculating 15 ml of H-media (10.00 g/L Bactopeptone, assay was 14.5 and 26.9 % respectively. In contrast, 3.00 g/L beef extract, 5.00 g/L NaCl, 1.00 g/L glucose) with a loop for cells treated kanamycin the percentage of adsorbed full of E. coli B23. The culture was incubated overnight at 37°C in an air shaker at 100 rpm. Phage bottom agar (PBA) plates (13.0 g/L phage after 4 and 7 minutes was 2.4 and 5.2, Bactotryptone, 8.0 g/L sodium chloride, 2.0 g/L sodium citrate, 1.3 respectively. The slope of the line for the kanamycin g/L glucose, 15.0 g/L agar, at pH 7.2) were warmed in a 37°C treated cells was -0.007 and the correlation between incubator 30 minutes prior to utilization. 2.5 ml aliquots of Phage Top variables was 0.83 (figure 1). In contrast, the slope of Agar (PTA) (13.0 g/L Bactotryptone, 8.0 g/L sodium chloride, 2.0 g/L sodium citrate, 3.0 g/L glucose, 7.5 g/L agar, at pH 7.2) were the line for the untreated cells was more than fivefold boiled at 100°C to melt and then kept at 48°C in a water bath. T7 steeper, with a value of -0.04 and a correlation of stock solution was serially diluted to 10-6, 10-7, 10-8 and 10-9. 200 μl of 0.99. The steeper slope for the untreated cells indicates overnight E. coli B23 culture and 200 μl of diluted phage were added greater phage adsorption for this sample than for the to 2.5 ml of liquid PTA at 48°C. Tubes were mixed and then poured onto warmed PBA plate. PTA solidified for 15 minutes at room kanamycin treated cells. The increase of unbound temperature and then the inverted plates were incubated at 37°C for phage to 102 % of the initial titre at 1 minute for the 18-24 hours. Plaques were counted and phage stock concentration kanamycin treated cells does not likely represent a true was determined. increase in the titre of phage, since progeny phage Growth of E. coli B23. An overnight culture was prepared by inoculating 15 ml of H-media with a loop full of E. coli B23. The would not have been produced within one minute. In next morning two flasks containing 25 ml of H-media were order to represent the fraction of unbound phage as inoculated with 200 μl of the overnight culture. The newly inoculated positive values, the P/Po ratio was multiplied by 10 cultures were grown for three hours at 37°C in an air shaker at 100 before taking the logarithm. rpm. Kanamycin monosulfate (Gibco-BRL#11815-024) was mixed with distilled water to give a final concentration of 20 mg/ml and then filter sterilized. 25 μl was then added to one of the 25 ml E. coli B23 to make a final antibiotic concentration of 20 μg/ml. The antibiotic treated culture was then incubated at 37°C in an air shaker at 100 rpm for 1 hour. After incubation a phage binding assay was performed on both the antibiotic treated sample and the control sample. Phage binding assay. A culture of indicator cells was prepared by inoculating 5 ml of H-media with a loop full of E. coli B23 the afternoon before the phage assay was to be performed. The culture was incubated overnight at 37°C in an air shaker at 100 rpm. Phage binding assay was performed according to the protocol outlined by Adams et al with some modifications (1). Each E. coli culture for the phage binding assay was diluted with H-media to an OD660 of 0.4. For the phage binding assay, 120 μl of 2 x 105 PFU/ml phage solution were added to 6 ml of diluted bacterial culture and were mixed. 1 ml samples were removed at 0, 1, 4, and 7 minutes and centrifuged in an Eppendorf 5415D centrifuge for 1 minute at 5000 x g in order to isolate the unadsorbed phage in the supernatant. Between time FIG. 1 Effect of kanamycin on the adsorption of T7 phage to E. points the bacterial-phage culture was kept in a 37°C 25 rpm shaking coli B23. Error bars indicate calculated standard error at each time water bath. To enumerate the unadsorbed phage in each sample, point. Data represents the average of three replicates. 10P: Tenfold either 20 μl or 100 μl of the supernatant was added to a test tube unbound phage at indicated time point; Po: Unbound phage at time 0. containing 2.5 ml of molten PTA (48°C) and 200 μl of E. coli Red squares: kanamycin treated cells; blue diamonds: untreated indicator cells. Each tube was then mixed by phage style mixing and cells poured over a pre-warmed PBA plate.